Abstract: :
Purpose:We have proposed that the severity of ocular irritationis dependent on the extent of acute ocular injury and that extentof injury can be used as a mechanistic basis for the developmentand validation of a robust, in vitro alternative to live animaltests using a 3-dimensional corneal equivalent system. The purposeof this study was to establish extended life-span human celllines by transfecting normal corneal epithelium and keratocyteswith the human telomerase reverse transcriptase (hTERT) andmeasure the extent of injury within corneal equivalents exposedto a range of ocular irritants differing in type and severity.Methods:Primary human epithelial cells grown in low calciumcontaining KGM media (Clonetics) and keratocytes grown in DMEMsupplemented with 10% fetal calf serum were infected with aviral vector containing the genes encoding hTERT and puromycinresistance. Infected cells were selected and surviving cellscloned and isolated. Cell lines and corneal equivalents werecharacterized by Karyotype analysis, TRAP assay, populationdoubling time, protein biochemistry and immunocytochemistry.Results:One human corneal epithelial cell line and four cornealfibroblast cell lines each having over 100 population doublingshave been isolated and fully characterized. The human epithelialcell line shows a normal 46, XY karyotype and positive TRAPassay for telomerase activity. Growth of cells in high calciumcontaining media results in growth arrest of confluent culturesindicating normal contact inhibition. The keratocyte cell lineshave a positive TRAP assay but show polyploidy, consistent withhuman corneal keratocytes obtained from donors 18 years andolder. Corneal equivalents comprised of corneal epithelium culturedon top of corneal fibroblasts embedded in a collagen matrixshow normal epithelial stratification and the formation of abasal cell, a wing cell and a superficial cell layer. Treatmentof corneal constructs with a slight, mild and severe irritantresulted in similar, yet more extensive, injury with slightirritants damaging the epithelium, mild irritants damaging theepithelium and anterior keratocytes and the severe irritantdamaging the entire construct.Conclusion:Treatment of cornealconstructs with a mild irritant resulted in similar, yet moreextensive, denudation of the corneal epithelium compared toan established ex vivo corneal model but did not show damageto the underlying corneal keratocytes.